Production of foods and drinks containing bifidobacteria

ABSTRACT

Foods and drinks containing bifidobacteria are produced by inoculating and cultivating bifidobacteria or a mixture of bifidobacteria and lactic acid bacteria in a medium containing  alpha -starch-transformed rice and bifidobacteria-fermentable sugars, and processing the resultant cultivated medium to produce foods and drinks containing bifidobacteria. The  alpha -starch-transformed rice is produced by cooking rice. The medium for cultivating bifidobacteria may contain milk, and cultivating may be carried out under aerobic conditions.

BACKGROUND OF THE INVENTION

This invention relates to a method for producing foods and drinkscontaining bifidobacteria.

Bifidobacterium predominates in intestinal bacterial flora of sucklinginfants, and is being watched with keen interest since it has aninfluence on the health of breast-fed infants.

There are many reports on the study of the physiological significance ofthis bacteria, clarifying (1) the inhibitory effect on putrefaction byputrefactive bacteria, (2) the inhibitory effect on production of toxicamines, (3) the digestive effect on human milk casein by the action ofphosphoprotein phosphatase, and (4) the effect of suppressing the growthof pathogenic bacteria by lowering intestinal pH following production oforganic acids such as lactic acid, acetic acid, and formic acid.

However, this Bifidobacterium favourable to infants is present in a verysmall amount in the intestines of bottle-fed infants, which isconsidered to be one of the causes for their susceptibility tointestinal diseases, greater than that of breast-fed infants.

Aimed at approximating the intestinal flora of bottle-fed infants tothat of breast-fed infants, an attempt has been made to produceBifidobacterium-containing powdered milk for infants and to modifypowdered milk for infants in such a manner as to be similar to amother's milk.

However, due to the problems as mentioned below, it has been difficultto practice an industrial cultivation of Bifidobacterium in a mediumconsisting of milk only.

That is, as compared with dairy lactic acid bacteria which are widelyused in processing milk, Bifidobacterium has the following problems:

(1) An industrial mass cultivation is difficult since Bifidobacteriumrequires strict anaerobic conditions for growth, and accordingly entailsa large equipment cost and requires high level cultivation techniques;

(2) The nutritional requirement for the cultivation is complicated andfastidious and therefore the bacteria do not substantially propagate ona pure cow's milk medium containing no growth promoting substance suchas yeast extract, peptone and the like; and

(3) Acetic acid, the main metabolic product of Bifidobacterium, ishighly stimulative, and therefore generally impairs the taste andflavour of foods and drinks containing the same.

We have found that bifidobacteria having the above mentioned propertiescan be prosperously propagated under the same aerobic culture conditionsas in the cultivation of dairy lactic acid bacteria in a mediumcontaining as the main component a pasty or milky product ofα-starch-transformed non-glutinous or glutionous rices, polished, whole,non-polished or powdered thereof, and further containing sugarsfermentable by Bifidobacterium, such as glucose, lactose, fructose,galactose and the like (the sugar may vary depending on the species ofBifidobacterium used), and that the cultivated product has a goodflavour since the fermentation product, acetic acid, well matches withthe flavour of cooked rice.

SUMMARY OF THE INVENTION

An object of this invention is to provide a method for producing foodsand drinks containing bifidobacteria, characterized by inoculating andcultivating bifidobacteria or bifidobacteria and lactic acid bacteria ina medium comprising a milky mixture containing rice treated for α-starchtransformation and sugars fermentable by bifidobacteria or furthercontaining milk components in addition to the above ingredients, andoptionally processing the cultivated product into a form suitable forfoods and drinks.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show graphs illustrating the state of cultivation with thepassage of the cultivation time.

FIG. 3 shows the results of the organoleptic evaluation of a productcultivated with bifidobacteria.

DETAILED EXPLANATION OF THE INVENTION

The above mentioned properties of bifidobacteria are illustrated by thefollowing experiments. Viable cell count expressed by count per ml wasmeasured in accordance with the method described in "J. Food Hyg. Soc.Japan" (Vol. 18, pp. 537-546, 1977). Titratable acidity was expressed byamount in ml of 0.1 N NaOH solution required to neutralize 10 ml ofsample.

Experiment 1

A culture medium for this Experiment was prepared by adding about 750 mlof water to 150 g each of washed unpolished-, whole-, and polished-non-glutinous rices, heating each mixture at 120° C. for 15 minutes,emulsifying it by a mixer, adding 30 g of lactose to it and finallytopping up to 1000 ml by the addition of water. A reconstituted skimmilk having a non-fat milk solid concentration of 15% was used as areference culture medium.

Each culture medium thus prepared was placed in a 2000 ml-conical flaskhaving a cotton plug, and was then sterilized at 120° C. for 20 minutes.After cooling, 3.0% of each starter of bifidobacteria previouslyprepared was inoculated in the respective medium, and was subjected to astatic culture at 37° C. for 24 hours. Acidity and viable cell countwere then measured, and the results are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________           Media                                                                         unpolished rice                                                                          whole rice polished rice                                           +          +          +                                                Bifido-                                                                              lactose    lactose    lactose    milk                                  bacteria                                                                             acidity                                                                           viable count                                                                         acidity                                                                           viable count                                                                         acidity                                                                           viable count                                                                         acidity                                                                           viable count                      __________________________________________________________________________    B. bifidium                                                                          8.3 6.8 × 10.sup.8                                                                 8.1 6.6 × 10.sup.8                                                                 7.2 5.6 × 10.sup.8                                                                 6.3 4.2 × 10.sup.8              YIT4005                                                                       B.bifidum E                                                                          7.2 5.0 × 10.sup.8                                                                 7.0 5.5 × 10.sup.8                                                                 6.8 4.2 × 10.sup.8                                                                 2.3 5.8 × 10.sup.5              B.breve Y                                                                            9.6 7.9 × 10.sup.8                                                                 9.4 7.1 × 10.sup.8                                                                 8.3 6.9 × 10.sup.8                                                                 3.3 3.1 × 10.sup.5              B.breve S                                                                            9.0 6.6 × 10.sup.8                                                                 9.1 6.3 × 10.sup.8                                                                 8.5 5.6 × 10.sup.8                                                                 2.5 4.2 × 10.sup.4              B.longum                                                                             5.8 4.1 × 10.sup.8                                                                 5.6 4.3 × 10.sup.8                                                                 5.0 4.0 × 10.sup.8                                                                 2.8 2.0 × 10.sup.4              ATCC-15707                                                                    B.adolescentis                                                                       5.5 3.9 × 10.sup.8                                                                 5.0 3.6 ×  10.sup.8                                                                4.4 2.5 × 10.sup.8                                                                 2.5 3.8 × 10.sup.4              B.infantis                                                                           4.8 1.4 × 10.sup.8                                                                 4.6 1.5 × 10.sup.8                                                                 3.8 8.9 × 10.sup.7                                                                 2.6 5.9 × 10.sup.4              __________________________________________________________________________     Note:                                                                         Starting Viable Cell Count: 8.5 × 10.sup.5 ˜ 1.3 ×          10.sup.7, Acidity: 1.3 ˜ 1.5                                       

In the reference milk medium, strains other than B. bifidum YIT 4005 (amutant of bifidobacteria having properties of propagating under aerobicconditions in a milk medium Deposit No. FERM-3372 at FermentationResearch Institute, Government Industrial Research, Ministry ofInternational Trade and Industry) were not totally propagated. On theother hand, in the respective milky culture medium containing rice asthe starting material, all the bifidobacteria were vigorouslypropagated.

According to other experiments wherein the concentration of cuturemedium was varied, it was proved that propagation was accelerated inproportion to the increase in the concentration of rice until theconcentration of rice reached 20%. If the concentration exceeds theabove value, starching occurs and accordingly amylase treatment isrequired. However, this treatment did not have a substantial influenceon the propagation.

With regard to unpolished-, whole-, and polished-glutinous rices,substantially the same results as in the above were obtained.

Experiment 2

The same procedures as in Experiment 1 were repeated, except that amedium used for this experiment was prepared by cooking unpolished-,whole-, and polished-non-glutinous rices, emulsifying it at aconcentration of 15%, and adding to the emulsion 2.0% of lactose and1.0-15% of skim milk powder.

Table 2 shows the results obtained with media containing skim milkpowder at a concentration of 5%. In the media containing milk componentsin addition to rice and sugar, the propagation of bifidobacteria wasaccelerated as compared with Experiment 1 wherein milk component was notused. The effect of the milk components on accelerating propagation wasenhanced in proportion to the increase in the concentration of skim milkpowder until the concentration reached 10%. If the concentrationexceeded the above value, no further enhancement of the effect onaccelerating propagation was achieved.

Substantially the same results are to be obtained by the use of wheypowder.

                                      TABLE 2                                     __________________________________________________________________________           rice                                                                   Bifido-                                                                              unpolished rice                                                                          whole rice polished rice                                    bacteria                                                                             acidity                                                                           viable count                                                                         acidity                                                                           viable count                                                                         acidity                                                                           viable count                                 __________________________________________________________________________    B.bifidum                                                                            13.5                                                                              6.2 × 10.sup.9                                                                 13.3                                                                              6.0 × 10.sup.9                                                                 12.6                                                                              5.8 × 10.sup.9                         YIT 4005                                                                      B.bifidum E                                                                          9.5 7.6 × 10.sup.8                                                                 9.6 7.0 × 10.sup.8                                                                 9.4 6.8 × 10.sup.8                         B.breve Y                                                                            11.8                                                                              5.0 × 10.sup.9                                                                 10.7                                                                              3.3 × 10.sup.9                                                                 10.1                                                                              3.4 × 10.sup.9                         B.breve S                                                                            10.0                                                                              9.0 × 10.sup.8                                                                 9.0 8.2 × 10.sup.8                                                                 9.1 7.8 × 10.sup.8                         B.longum                                                                             7.9 6.7 × 10.sup.8                                                                 7.6 5.8 × 10.sup.8                                                                 7.2 5.3 × 10.sup.8                         ATCC-15707                                                                    B.adolescentis                                                                       6.5 5.1 × 10.sup.8                                                                 6.3 5.0 × 10.sup.8                                                                 6.0 4.1 × 10.sup.8                         B.infantis                                                                           6.6 4.9 × 10.sup.8                                                                 6.0 4.1 × 10.sup.8                                                                 5.6 3.9 × 10.sup.8                         __________________________________________________________________________     Note:                                                                         Starting Viable Cell Count: 1.0 ˜ 3.4 × 10.sup.7, Acidity: 1.     ˜ 2.0                                                              

Experiment 3

Two species of bifidobacteria or a mixture of bifidobacteria and lacticacid bacteria were inoculated in a milky medium prepared in such amanner as to contain 15% of unpolished non-glutinous rice, 5% of skimmilk powder and 2% of lactose, and was subjected to a static culture at37° C. The inoculum of the starter of each strain was respectively 2%.FIGS. 1 and 2 show the results of the acidity and viable cell countsmeasured with the passage of time during cultivation in comparison withthe results obtained by single cutivation of each strain. The strainsused in this Experiment are as follows:

B₁ : B. bifidum YIT 4005

B₂ : B. breve Y

LA: Lactobacillus acidophilus

As can be seen from the two Figures, a certain species of strain wasvigorously propagated in a rice-containing medium when subjected to amixed cultivation.

Experiment 4

B. bifidum YIT 4005 was inoculated in a medium containing 15% of wholerice, 5% of skim milk powder and 2.0% of lactose and in a skim milkmedium having a non-fat milk solid concentration of 15%, and wascultivated at 37° C. for 24 hours. With regard to the culture thusobtained, organoleptic evaluation was made by a panel consisting of 10trained panelists. The results are shown in FIG. 3.

Both cultures had almost equal values each other with regard to acidityand ratio of acetic acid/lactic acid (see Table 3). However, culture ofthe rice-containing medium was evaluated to have a better flavour sincethe flavour of cooked rice well matches with acetic acid and thestimulative smell of acetic acid is weaker as compared with the productcultivated in the milk-containing medium.

                  TABLE 3                                                         ______________________________________                                                       Culture                                                                                   Acetic Acid/                                       Medium           Acidity   Lactic Acid                                        ______________________________________                                        medium consisting                                                                              6.8       1.75                                               of milk only                                                                  medium containing                                                                              7.5       1.80                                               rice                                                                          ______________________________________                                    

The present invention was accomplished in view of the above knowledge.Thus, the present invention resides in a method for producing foods anddrinks containing bifidobacteria, characterized by inoculating andcultivating bifidobacteria or bifidobacteria and lactic acid bacteria ina medium comprising a milky mixture containing rice treated for α-starchtransformation and sugars fermentable by bifidobacteria or furthercontaining milk components in addition to the above ingredients, andoptionally processing the cultivated product into a form suitable forfoods and drinks.

As clearly described above, any glutinous or non-glutinous rice can beused in the method of this invention, and the rice may or may not, bepolished or milled, to any degree. When these rices are used in a mediumfor cultivating bifidobacteria, they are preferably made into ahomogenous milky state by the method used in Experiment 1 or a method ofgrinding and heating in water. An appropriate concentration of rice in amedium is 10-20%, preferably 15%.

Sugars such as lactose, fructose and glucose are the most preferablebifidobacteria-fermentable sugars since they are easily available andfermentable by all of the bifidobacteria. It is not necessary to usepure sugars, but any edible material containing these fermentablesugars, for example, lactose-containing milk, skim milk, whey and thelike can be used as a medium component. Milk components are particularlypreferable since they accelerate the propagation of bifidobacteria andprovide a product having a good nutrition balance. A part or the wholepart of the rice may be previously treated with amylase or withamylase-producing Aspergillus oryzae, Aspergillus niger, Bacillussubtilis and the like to produce glucose in an amount sufficient for thepropagation of bifidobacteria, and the glucose thus produced may be usedas a fermentable sugar for bifidobacteria. This method has advantages inthat the emulsification of rice proceeds along with saccharification,and that a unique flavour can be obtained.

The concentration of bifidobacteria-fermentable sugars in a mediumshould preferably be 1-5%, more preferably about 3%.

Any cultivation suppliments or seasonings, spices, nutritions and thelike may be added to a medium in addition to the above components.

Bifidobacteria to be inoculated in the above mentioned homogenous milkymedium are not specially limited, and 2 species or more ofbifidobacteria may be used. In combination with bifidobacteria, Lacticacid bacteria may be inoculated, preferable examples of which includeLactobacillus acidophilus, L. casei, L. bulgaricus, Streptococcusthermophilus and the like.

The cultivation of bifidobacteria can be carried out under aerobicconditions in the same manner as in the cultivation of ordinary lacticacid bacteria, and strictly anaerobic conditions are not required. Thisis a great advantage of this invention, and the conventional apparatusand techniques used in the cultivation of lactic acid bacteria can beused as they are.

Under normal cultivation conditions, viable cell count reaches maximumafter 18-24 hour cultivation, and pH is gradually decreased because acidis formed along with the passage of the cultivation time. The productionof acetic acid by bifidobacteria starts at the initiation ofcultivation. If the cultivation is further continued, viable cell countstarts to be decreased after 24 hours and the production of acid iscontinued for some time but the production of acid stops to some extent.Thus, taking the use of the cultivated product into consideration,cultivation is stopped when the pH of the medium reaches 4.2 to 5.6. Inmost cases using bifidobacteria per se, the viable cell count ofbifidobacteria should preferably be more than 10⁷ per ml. According tothe culture method of this invention, it is quite easy to obtain aviable cell count of 10⁸⁻⁹ /ml.

The product of this invention contains acetic acid 80-120 mM/l andlactic acid 50-70 mM/l as the main metabolites in addition to the viablebifidobacteria. The contents of these acids are not specially differentfrom those in conventional bifidobacteria culture, but the acetic acidwell matches with emulsified rice and therefore the cultivated productof this invention deserves much higher organoleptic evaluation than theconventional cultivated products. Thus, the cultivated product of thisinvention can be served, as it is, or as a food containing viablebifidobacteria without flavouring. The product of this invention may beserved as a drink by optionally adding sweetening materials, fruitjuice, water, spices or the like to modulate the concentration andflavour. The product can also be served as powdery or tablet-like foodsor medicinal preparations containing viable bifidobacteria by drying. Aslong as the bifidobacteria do not perish, any conventional processingtechniques and any processed forms can be used.

The present invention is further illustrated by the following Examples.

EXAMPLE 1

To 15 Kg of fully washed polished non-glutinous rice, was added 70liters of water, and the resultant mixture was heated at 121° C. for 15minutes. The mixture was then emulsified in a mixer, and the mixture wastopped up to 100 liters by the addition of water. To this mixture, wasadded 2 kg of skim milk powder, and the mixture was sterilized at 121°C. for 40 minutes. The mixture was then cooled to 37° C. while stirring.

Into the milky rice medium thus prepared, 3% by volume of the starter ofB. bifidum YIT-4005 was inoculated, and was cultivated at 37° C. for 24hours.

After cultivation, the culture was homogenized in a homogenizer (150Kg/cm²), and 40 liters of syrup containing 4.8 Kg of sucrose was mixedtherewith, thus producing a drink having an acidity of 5.4 andcontaining bifidobacteria in a count of 4.3×10⁸ /ml. The product thusobtained had a less stimulative smell and provided a satisfactory tasteand flavour.

EXAMPLE 2

To 15 Kg of thoroughly washed whole glutinous rice, was added 70 litersof water, and the mixture was heated at 121° l C. for 15 minutes. Inorder to facilitate emulsification, 50 g of liquifying amylase (70,000units/g) was added to the mixture, and the mixture was emulsified in amixer. To this mixture, 1 Kg of lactose and 10 liters of cow's milk wereadded, and the mixture was topped up to 100 liters by the addition ofwater. The mixture was then sterilized at 121° C. for 40 minutes, andwas cooled to 37° C. while stirring. Into the medium thus prepared, 5%of the starter of B. adolescentis was inoculated, and was cultivated at37° C. for 40 hours.

After cultivation, the culture was treated in the same manner as inExample 1 to obtain a drink having an acidity of 6.1 and containingviable bifidobacteria in an amount of 8.0×10⁷ /ml.

Example 3

To 2 Kg of thoroughly washed polished non-glutinous rice, was added 7liters of water, and the mixture was heated at 121° C. for 20 minutes.After cooling, 200 g of seed starter of Aspergillus oryzae was added tothe mixture, and the mixture was shaken at 37° C. for 40 hours tosaccharify a part of the rice, thus forming glucose. Thereafter, themixture was emulsified in a mixer, and was topped up to 10 liters. Tothe emulsion thus prepared, was added 5 liters of syrup containing 600 gof sucrose and 30 g of gelatin, and the resultant mixture wassterilized. Into the medium thus prepared, 2% each of the starter of B.breve Y and B. longum (ATCC-15707) was inoculated for culture at 37° C.for 12 hours to obtain a sweetwine flavoured yoghurt-like food. Theproduct thus obtained had an acidity of 5.2, and the viable cell countwas 2.2×10⁸ /ml for B. breve Y and 7.8×10⁷ /ml for B. longum.

Example 4

One Kg of polished nonglutinous rice was thoroughly washed and dried.The rice was then powdered by a grinder, and 5 liters of water was addedto the powdered rice. The mixture was then heated at 121° C. for 15minutes. After cooling the mixture to 55° C., 2 g of saccharifyingamylase (50,000 units/g) was added to the mixture while stirringthoroughly, and the resultant mixture was treated at 55° C. for 1 hour.After the amylase-treatment, the mixture was topped up to 10 liters bythe addition of water, and was sterilized. After cooling to 37° C., themixture was inoculated with the starter of Saccharomyces sake, and wascultivated at 30° C. for 20 hours. Thereafter, the culture medium thusprepared was sterilized again, and 2% each of the starter of B. bifidumE and Lactibacillus acidophilus was inoculated into the medium. Thecultivation was carried out at 37° C. for 30 hours.

After cultivation, the culture was homogenized in a homogenizer, and 2liters of syrup containing 300 g of sucrose was added to the culture.The product thus obtained contained a small amount of alcohol, and had atitrarable acidity of 9.6 and the viable cell count of 1.5×10⁸ /ml forbifidobacteria and 6.6×10⁷ /ml for lactobacilli.

Example 5

To the culture prepared using the same materials in the same manner asin Example 1, were added 5 Kg of skim milk powder, 5 Kg of sugar and 1Kg of Vitamin C, and the mixture was dried by a spray drier, thusproducing 26.5 Kg of powdery food containing 7.1×10⁸ /g ofbifidobacteria.

What we claim is:
 1. A method of producing foods and drinks containingbifidobacteria by inoculating and cultivating bifidobacteria or amixture of bifidobacteria and lactic acid bacteria in a mediumconsisting essentially of 10 to 20 percent by weightα-starch-transformed rice, which rice has been transformed by cooking,and bifidobacteria-fermentable sugars in an amount of 1-5 percent of thetotal weight of the medium to produce a bifidobacteria-containing mediumhaving a bifidobacteria cell count of at least 10⁷ cells/ml., andpreparing a food or drink from said bifidobacteria-containing medium. 2.A method according to claim 1, in which the medium contains two speciesor more of viable bifidobacteria.
 3. A method according to claim 1wherein said medium further consists essentially of milk in an amount of1-15 percent by weight.
 4. A method according to claim 1 whereinsweetening materials, fruit juices or spices are added to thebifidobacteria-containing medium.
 5. A method according to claim 1wherein said bifidobacteria-fermentable sugar is at least one selectedfrom the group consisting of glucose, lactose, fructose, galactose andmixtures thereof.
 6. A method according to claim 1 wherein thebifidobacteria-containing medium is dried to produce a dried product. 7.A method according to claim 1 in which cultivation is stopped when thepH of the medium is between 4.2 to 5.6.
 8. A method according to claim 1in which the cultivation is carried out under aerobic conditions.
 9. Afood or drink prepared from the bifidobacteria-containing mediumproduced by the method of claim 1, 2, 3, 4, 5, 6, 7, or 8.